In our previous genome-wide scan searching for type 2 diabetes mellitus (T2DM) genes in the Pima Indians, the most significant linkage was found on Chromosome 1q21-q23. Subsequently, the same region has been linked with T2DM in four different Caucasian groups, thus providing a replication of our finding consistent with an important diabetes susceptibility locus in this genomic interval. Our search for the underlying gene involves analyses of densely spaced single nucleotide polymorphisms (SNPs) in selected affected an unaffected Pimas for associations with the disease to narrow the likely interval harboring the susceptibility locus. A high SNP density must be achieved for an efficient search for the T2DM gene and our current goal is to analyze one SNP every 50 kb throughout the linked region. As a complementary approach, we are also investigating relevant candidate genes within this interval. To increase the efficiency of SNP analysis, we have been utilizing a pooled DNA approach (i.e. estimating allele frequency differences between diabetic and control groups by typing SNPs in pooled DNA samples representing the respective groups). The pooled genotyping has been performed by mass spectrometry at Sequenom in San Diego, using over 1000 SNPs, which we selected from public databases. Based on the pooling results, we then genotype in every individual any SNPs indicating significant differences between the pools. Several of the SNPs typed at Sequenom showed significant differences between the diabetic and control pools, but we were not able to reproduce these results by typing these markers in the individuals. Based on our data, one of the most likely explanations for these discrepancies is an insufficient sensitivity of the technique. Because DNA pooling is potentially a very efficient and cost-saving approach in positional cloning, we are now exploring other, more sensitive techniques (Pyrosequencing) for this purpose. Simultaneously, we continue typing markers individually to assess their association with T2DM. We expect that this strategy will help to define the critical interval harboring the susceptibility locus that will aid in the selection of candidate genes for mutation screening in diabetic subjects.